Colored Overlay Dot Plots and Backgating

A useful tool in the analysis of cytometry data is the ability to overlay plots that would otherwise be viewed separately. The overlaid plots can be colored according to different gated populations or samples within an experiment.

(pSTAT3 expression is compared between different populations gated from PBMC stimulated with IL6 and IL10. Separate plots can be easily converted into a colored overlay for a different visualization)

In Cytobank, any figure dimension can be used to create a colored overlay figure. See how timepoints stack up or compare patients or doses. Overlay populations to backgate for immunophenotyping or to visualize small populations of cells among a greater overall background.

(A rare signaling population is visualized in a larger context.)

Pair colored overlay dot plots with viSNE to inform the quality of a manual gating strategy by comparing the location of colored events from gated populations to their location on top of a viSNE map.

(An incomplete collection of manually gated populations are overlaid on the corresponding viSNE map of CD45+ PBMC (“Ungated” after viSNE). Blue events were not captured by manual gates, showing shortcomings in the gating strategy)

How to Make a Colored Overlay Dot Plot

To create a colored dot overlay, navigate to the working illustration and select a plot type of dot with a coloring mode of overlaid figure dimension. Note that this will transform the label of the second figure dimension to say “Overlaid” instead of "Rows". The colored components of each overlay are determined by the selections within the second (Overlaid) figure dimension. The order of the variables within the second figure dimension determine the stacking order and colors of the overlay. The top most variable forms the base layer of the overlay. To change the overlay ordering, open the figure dimension, then click and drag to vertically reorder the variables. The dot size and dot shape can be configured. Colors cannot currently be adjusted.

(The working illustration configuration for colored dot plot overlays. As is the case with other dot plots within Cytobank, the size and shape of each dot can be configured. Other relevant controls are shown, such as the X and Y axis settings, and the compensation selector)

Number of Colors and Customizing Colors

There are 64 available colors within a colored overlay that have a set order and color.

Currently, dot overlay colors cannot be customized within Cytobank. Color customization can be done simply by importing the image into a separate software and doing a color swap.

Get in touch with Cytobank Support if you need more colors or want to set custom colors.

Video Introduction to Colored Overlay Dot Plots

(video introduction to colored overlay dot plots. Note that styling of the Cytobank interface within the video is outdated, but the same workflow can be accomplished very similarly with current styling)